Part:BBa_K3185005

SPYCatcher -> CenA W68Y

Usage and Biology

CBM-CenA is a Carbohydrate-binding Module from cellulase of Cellulomonas fimi. Originally, it has binding affinity to cellulose, but its binding ability to PET is enhanced because of point mutation[1]. In the paper, they compare many kinds of mutants and it is found that a mutant whose #68 amino acid W is changed by Y (CenA W68Y) has the most strong binding affinity to PET.

We used it for PET binding domain. We put SpyCatcher on the N-terminus of CenA W68Y because we used SpyTag/SpyCatcher system to bind it to other parts. In addition, this has three tag and cleavage sites. First is inserted 6×His-tag in N-terminus of SpyC to purify. Second is inserted MYC-tag between SpyCather and CBM(Reference) to detect it by using the antibody. Third is inserted TEV protease site because, in the paper, it is used for protein purification. However, we didn’t use it in our experiment.

We put it between the BamHI site and the Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used the Ni-NTA agarose for purification. After that, we confirmed the molecular weight of SpyCatcher-CenA by using SDS-PAGE.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 751
  Illegal AgeI site found at 841
  
• 1000
  COMPATIBLE WITH RFC[1000]

Purification

Expression

• Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37^oC shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer.

• Protein was expressed in 0.1mM IPTG for 2hours.

Refrences

[1](Zhang et al. 2013/ Zhang et al. 2010)